title: Improved detection of rare HIV-1 variants using 454 pyrosequencing.
abstract: 454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.
authors.affiliation: Department of Microbiology, University of Washington, Seattle, Washington, United States of America.
authors.forename: Brendan B
authors.initials: BB
authors.lastname: Larsen
authors.forename: Lennie
authors.initials: L
authors.lastname: Chen
authors.forename: Brandon S
authors.initials: BS
authors.lastname: Maust
authors.forename: Moon
authors.initials: M
authors.lastname: Kim
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: Wenjie
authors.initials: W
authors.lastname: Deng
authors.forename: Dylan
authors.initials: D
authors.lastname: Westfall
authors.forename: Ingrid
authors.initials: I
authors.lastname: Beck
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Frenkel
authors.forename: James I
authors.initials: JI
authors.lastname: Mullins
mesh_terms: D016679:Genome, Viral; D015658:HIV Infections / Q000175:diagnosis / Q000635:transmission* / Q000821:virology*; D015497:HIV-1 / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity